Xiao-Hong Liu

Synergistic effect of fibronectin and hepatocyte growth factor on stable cell-matrix adhesion, re-endothelialization, and reconstitution in developing tissue-engineered heart valves.

Stable cell-matrix adhesion, re-endothelialization, and reconstitution represent important issues in creating autologous living heart valve, a close collaboration between growth factors and the extracellular matrix in these processes appears crucial. To prove this action, porcine decellularized valve constructs were precoated with fibronectin and seeded with hepatocyte growth factor-transferred marrow stromal cells (MSCs) and grown in vitro in a pulsatile-flow bioreactor. Results showed hepatocyte growth factor stimulated adhesion of MSCs to fibronectin in a time-dependent manner with a range of 8–128 ng/ml. Histological observation demonstrated a time course of MSC growth on decellularized valve constructs. A handful of cells, a loose cellular layer, a confluent monolayer coverage, a 2-layer structure and a 3-layer structure were observed at weeks 2, 3, 4, 6, and 8, respectively. Immunohistochemical analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor positive) and myofibroblasts (α-smooth muscle actin and vimentin double-positive) at week 8. Importantly, endothelial cell retention (17.3 ± 2.6/mm) remained high under exposure to high flow and pressure conditions in a bioreactor. These results demonstrated that the combination of fibronectin and hepatocyte growth factor contributed to creating autologous living heart valve.

Regional expression of the hypoxia-inducible factor (HIF) system and association with cardiomyocyte cell cycle re-entry after myocardial infarction in rats

Hypoxia-inducible factor (HIF)-1α and-2α have diverse actions on the myocardium, but the importance of direct effects on cardiac myocytes is unclear. To define their regional accumulation and association with cardiomyocyte cell cycle change after myocardial infarction (MI), a rat MI model was established by occluding the coronary arteries. To further prove a causative relationship between HIF and cell cycle regulation, cultured cardiomyocytes were transfected with adenoviral vectors carrying HIF-1α and HIF-2α. Two weeks after MI, both HIF-1α and HIF-2α mRNA were moderately increased in the infarcted left ventricle and noninfarcted left ventricle; HIF-2α amplification was also detected in areas of the interventricular septum and the right ventricle. In concordance with the changes in mRNA levels, immunohistochemistry signals of HIF-1α and HIF-2α were characterized by different regional distributions. In the myocardium adjacent to the infarcted tissue, a significant correlation between HIF-1α or HIF-2α and Ki-67 labeling index was observed (P < 0.001). Immunohistochemical double staining showed that HIF positive cardiomyocytes underwent DNA synthesis. Cardiomyocytes treated with HIF-1α or -2α expressed Ki-67, phosphohistone H3, and bromodeoxyuridine effectively in vitro. In conclusion, HIF-1α and HIF-2α had a distinct spatial expression pattern in a rat model of ischemic heart disease. Both HIF subunits might be potent stimuli for cardiomyocytes to re-enter the cell cycle and initiate DNA synthesis.

Expression of stem cell factor in gastrointestinal stromal tumors: Implications for proliferation and imatinib resistance

KIT autophosphorylation caused by mutation of KIT is considered to be a critical mechanism for the oncogenesis of gastrointestinal stromal tumors (GISTs). However, little is known regarding whether stem cell factor (SCF), the KIT ligand, is able to induce the proliferation of GIST cells by activating the wild-type KIT receptor in GISTs. Imatinib, a tyrosine kinase inhibitor, has been demonstrated to be effective as treatment for the majority of GISTs. However, primary resistance to imatinib in GISTs with wild-type KIT and acquired resistance in GISTs with mutant KIT are becoming increasingly significant problems. The aims of this study were to detect the expression and function of SCF in 68 GIST samples, and to explore the relationship between SCF activity and imatinib resistance using immunohistochemical staining and western blot analysis. Results showed abundant expression of SCF in GISTs and demonstrated that SCF is capable of enhancing GIST cell proliferation. Similar to its ineffectiveness in wild-type GISTs, imatinib also failed to inhibit SCF-induced KIT activation in GISTs with mutant KIT. We also found increased SCF expression in GIST cells treated with imatinib. Overall, our results indicated that SCF-induced KIT activation is a novel essential pathway for the proliferation of GISTs. Imatinib was not able to inhibit the activity of SCF, while it promoted the expression of SCF, which may have contributed to acquired imatinib resistance.

Expression profiles of stemness genes in gastrointestinal stromal tumor

Gastrointestinal stromal tumor (GIST) is believed to originate from intestinal cells of Cajal or their stem cell precursors, and expresses stemness-related markers, such as CD117, CD34, DOG1 and nestin. To further characterize phenotypic features of GISTs, we examined expression profiles of a panel of stemness genes in GISTs, by analyzing existing gene expression profiling datasets. Our results showed that mRNA levels of B-lymphoma moloney murine leukaemia virus insertion region-1 (BMI1), kruppel-like factor 4 (KLF4), sal-like protein 4 (SALL4) and telomerase reverse transcriptase (TERT) were significantly unregulated in GISTs. Subsequently, protein expression of BMI1 and TERT was identified in GIST specimens by immunohistochemistry. Especially, we found that high expression of nuclear BMI1 was associated with large tumor size (P = .0239), high mitotic count (P < .01), high Ki-67 index (P = .0357), advanced National Institute of Health (NIH) criteria (P = .0025) and advanced World Health Organization (WHO) classification (P < .01) in GISTs. Functional and pathway enrichment analysis showed that most of BMI1s coexpressed genes were involved in tumor growth-related process, such as regulation of cell cycle and proliferation. Furthermore, we confirmed RAS oncogene family (RAB18) and limb development membrane protein 1 (LMBR1) genes as novel targets for BMI1 in GIST cells. These results provide valuable information for the expression profiles of stemness genes in GISTs, and identified nuclear BMI1 as an important marker of GIST cell proliferation and progression.

FoxM1 is regulated by both HIF-1α and HIF-2α and contributes to gastrointestinal stromal tumor progression

BackgroundFoxM1 plays important regulatory roles in a variety of diseases. However, the functional role of FoxM1 and mechanisms responsible for its expression in gastrointestinal stromal tumor (GIST) is not thoroughly understood.MethodsFoxM1 protein expression and biological function were examined in human GIST tissues and cells using immunohistochemistry, quantitative real-time PCR, western blot, CCK-8, wound-healing- and Matrigel invasion assays, respectively. The role of hypoxia-inducible factor (HIF) signaling in FoxM1 expression was investigated using chromatin immunoprecipitation and luciferase reporter and in vivo tumor growth assays.ResultsFoxM1 was highly expressed in highly proliferative and migratory/invasive GIST specimens. Upregulation of FoxM1 was positively correlated with the expression of HIF-1α and HIF-2α in GIST specimens, and hypoxia-induced FoxM1 expression in GIST cells. Functionally, ectopic expression of FoxM1 significantly promoted GIST cell proliferation, cell cycle progression, migration and invasion, whereas the knockdown of endogenous FoxM1 of hypoxic GIST cells had the opposite effects. Molecularly, FoxM1 was transcriptionally regulated by HIF-2α under normoxia, whereas it was upregulated by both HIF-1α and HIF-2α under hypoxia. The xenograft tumor data further confirmed the regulated effect of HIF-1α and HIF-2α on FoxM1, and demonstrated that the simultaneous downregulation of both HIF-1α and HIF-2α inhibited GIST tumor growth.ConclusionsOur data demonstrated the critical role of FoxM1 in promoting GIST progression and uncovered a novel HIF-1α/HIF-2α-FoxM1 axis. These findings identify FoxM1 as a possible new molecular target for designing novel therapeutic treatments to control GIST progression.